My Total RNA degraded? - (Jul/08/2006 )

Hi everyoe:

Yesterday, I isolated Total RNA from Human Skin with Trozl, then I did electrophoresis to detect the Total RNA quantity, (The Electrophoresis Image is attached below). The 18S bands is so weak, While 28S band is so bright, A260 value of the Total RNA show that, the total RNA is 0.34ug/ul, the Sample I load on the agarose gel is 6ul,namely about 2ug. ( RNA marker size: from up to low is 6kb,4kb,3kb,2kb,1.5kb) I wonder whether Total RNA is degraded by RNase? Any one has any ideas?

By the Way, any one knows what tissues Human TypeI Collagen is highly expressed besides skin?

Thanks in advance!

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Redo extraction and all your material and centif tube mus be double autoclaved.
your RNA is starting degradation .

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Hi Najib:

Thanks for your patient reply and your kind suggestion!

Anyone has more suggestion?!

BTW, Is 5mM Mg2+ is a bit high in Reverse transcription of mRNA longer than 4kb? What Mg2+ concentration is optimizing to a 5.5kb mRNA with 65%GC Content with Superscript II from Invitrogen due to your bench experiences?!

Thanks in advance!

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well, basics : work quick in clean bench area, and at lower temp (4° most of time is good).
your RNA is degraded.
I've learned that collagen I was xpressed in bones derma and tendon (sorry if that wasn't your question...)

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